Mutated factor x polypeptides and uses thereof for the treatment of haemophilia

ABSTRACT

The present invention relates to mutated factor (FX) polypeptides and uses thereof for the treatment of haemophilia. In particular, the present invention relates to a mutated factor X (FX) polypeptide wherein the heavy chain comprises at least one mutation selected from the group consisting of: —the mutation which consists of the substitution of the glutamic acid residue (E) at position 255 of Seq. ID No. 1 by a glutamine residue (Q), an asparagine residue (N), a serine residue (S), an alanine residue (A), or a tyrosine residue (Y); —the mutation which consists of the substitution of the glutamic acid residue (E) at position 256 of Seq. ID No. 1 by a glutamine residue (Q); and —the mutation which consists of the substitution of the glutamic acid residue (E) at position 258 of Seq. ID No. 1 by a glutamine residue (Q);

FIELD OF THE INVENTION

The present invention relates to mutated factor (FX) polypeptides anduses thereof for the treatment of haemophilia.

BACKGROUND OF THE INVENTION

Haemophilia A and B are inherited X-linked coagulopathies characterizedby a lack of coagulation factor VIII (FVIII) and factor IX (FIX)respectively. Haemophilia A affects about 1 in 5 000 and Haemophilia Babout 1 in 35 000 male births (1,2). Haemophilia leads to impairedthrombin generation, weak and vulnerable clots, and thereforespontaneous bleeding. Compared to severe patients, bleeding episodes areless frequent in moderate/mild patients, and are usually provoked bytrauma or invasive procedures. Current treatment includes replacementtherapy with recombinant or purified FVIII or FIX (3). However, one ofthe most serious drawbacks of these treatments is the development ofalloantibodies, called inhibitors, inhibiting the activity of thecoagulation factor, in a non-negligible number of patients withhaemophilia A: 20-25% of severe patients and 7-13% of moderate/mildpatients (4). Treatment of these inhibitor patients is limited to FVIII-or IX-bypassing agents, such as recombinant FVIIa (Novoseven®) orplasma-derived activated prothrombin complex (FEIBA®). However, theseproducts have considerably shorter half-lives (4-7 h for FEIBA® and1.5-2.7 h for Novoseven®), than the respective half-lives of FVIII (˜12h) and FIX (˜18 h). These short half-lives require the need for frequentinfusions, limiting the use of both products for prophylactic purposesand increasing the costs. They are expensive and a substantial number ofpatients do not respond to these agents.

Therefore, as an alternative therapeutic approaches, Factor X (FX) is amore attractive bypassing molecule, since it displays a 40 h half-lifeand is part of the coagulation cascade normally activated by both FVIIIand FIX. For instance, a FX variant which combines activation of themolecule by thrombin with a long survival mimicking that of the FXzymogen was disclosed in WO 2010070137. This FX variant is activatedwithout FVIII or FIX, in vitro as well as in vivo and could thus be usedas a bypassing agent in both hemophilia A and B. However, the FXvariants of the prior art are susceptible to inactivation due to thepresence of inhibitors such as Antithrombin (AT) and/or the TissuePathway Inhibitor (TFPI). Actually, TFPI in complex with FXa (TFPI)-FXainhibits the initiating complex of the coagulation Tissue Factor(TF)/FVIIa. TF/FVIIa complex initiates coagulation by activating FX.However, activation of FX to FXa by the FVIIIa/FIXa complex counteractsthe activity of TFPI, allowing coagulation to proceed. In patients withhaemophilia, the propagation phase of thrombin generation is notsustainable due to the lack of intrinsic tenase (FVIIIa/FIXa) complex(11). The limited availability of thrombin via TF-FVIIa pathway is inpart due to rapid inhibition of FXa and thrombin by TFPI and AT (12,13). In particular, plasma AT levels are normally high (2.6 μM), and ATis capable of inhibiting trace amounts of FXa and thrombin (13, 14).Therefore, a new upcoming strategy to treat coagulation disorders likehemophilia has recently emerged and implies neutralization of naturalanticoagulants and especially TFPI and AT. This is based on severalobservations. Thus, low TFPI and AT levels in neonates are deemed to beimportant in augmenting thrombin generation with lower levels ofprocoagulant factors (15, 16). Furthermore, low AT levels improvedhaemostatic function in FVIII-deficient mice with heterozygous ATdeficiency (17). Very recently, a biopharmaceutical company (Alnylam)developed an RNAi therapeutic targeting antithrombin (ALN-AT3) for thetreatment of haemophilia and other rare bleeding disorders, which iscurrently being investigated in a multinational Phase 1 trial inhaemophilia subjects. However, because of the high concentration ofcirculating plasma antithrombin (3-5 μM), targeting antithrombin viainhibitory molecules may not be the ideal way to obtain therapeuticattenuation of coagulation in haemophilia patients. For TFPI, differentblocking reagents have been evaluated as possible therapeutic agents indifferent animal models with haemophilia (18, 19, 20, 21). However,there are some drawbacks for anti-TFPI agents. Indeed, TFPI isdistributed among different pools: the major part is located in or atthe endothelial surface, while the rest is distributed equally betweenplatelets and plasma. Moreover, only 1% of total TFPI circulates as freeprotein in plasma, with the remainder bound to LDL-particles. Due tothis complex biodistribution, it is difficult to monitor the efficacy ofTFPI inhibition upon treatment in patient plasma samples. A second issueis that different splicing forms of TFPI are present (TFPIalpha andTFPIbeta), which act in different ways. Anti-TFPI based therapy shouldtherefore be using agents that differentiate between both forms. Toillustrate the difficulty to use the TFPI-blocking strategy, a clinicaltrial ran by Baxter using an aptamer directed against TFPI has beenstopped due to an increased number of bleeding events. Biologicalexplanations for this observation is that the blocking agent releasesintracellularly stored TFPI, impacts its metabolism, and prolongs itscirculatory half-life.

SUMMARY OF THE INVENTION

The present invention relates to mutated factor (FX) polypeptides anduses thereof for the treatment of haemophilia. In particular, thepresent invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

The inventors now describe new FX polypeptides in which residues in theheavy chain were mutated at positions 215, 216, 218 from Glu (E)(FIG. 1) to Gln (Q). These selectively mutated residues on FX could thusinterfere with the inhibition, when activated, by Antithrombin (AT)and/or the Tissue Pathway Inhibitor (TFPI). The inventors demonstratethat these molecules have the capacity to correct for the absence ofFVIII or FIX in a calibrated thrombin generation assay with or withoutinhibitor. These properties give the proteins the potential to be usedas bypassing agents in both haemophilia A and B, either episodically orprophylactically, and irrespective of the presence of inhibitors sincecalibrated thrombin generation assay has been shown to predict theresponse to bypassing agents (22).

A first object of the present invention relates to a mutated factor X(FX) polypeptide wherein the heavy chain comprises at least one mutationselected from the group consisting of:

-   -   the mutation which consists of the substitution of the glutamic        acid residue (E) at position 215 by a glutamine residue (Q), an        asparagine residue (N), a serine residue (S), an alanine residue        (A), or a tyrosine residue (Y);    -   the mutation which consists of the substitution of the glutamic        acid residue (E) at position 216 by a glutamine residue (Q); and    -   the mutation which consists of the substitution of the glutamic        acid residue (E) at position 218 by a glutamine residue (Q);

As used herein, the term “factor X” has its general meaning in the artand refers to a secreted serine protease implicated in coagulationmechanisms. Typically, the amino acid sequence of the human factor X isprovided by SEQ ID NO:1 (FIG. 1). The numbering systems used to localizethe amino acid residues for factor X is based on the sequence deducedfrom the secreted protein, which contains the light chain, theactivation peptide and the heavy chain: the amino acid residue numbered1 is the first amino acid residue of the amino-terminal extremity of thelight chain. As shown in FIG. 1, the sequence of factor X is divided infive different regions, which correspond, according to the usednumbering system to:

-   -   the pre-peptide (or signal peptide) between the positions −40 to        −28,    -   the pro-peptide between the positions −27 to −1,    -   the light chain between the positions 1 to 142    -   the activation peptide between the positions 143 to 194    -   the heavy chain between the position 195 to 448.

The signal peptide is cleaved off by signal peptidase, the propeptidesequence is cleaved off after gamma carboxylation took place at thefirst 11 glutamic acid residues at the N-terminus of the matureN-terminal chain. A further processing step occurs by cleavage betweenArg142 and Ser143 according to the used numbering system (FIG. 1,positions 182-183 of SEQ ID NO:1). This processing step also leadsconcomitantly to the deletion of the tripeptide Arg140-Lys141-Arg142(positions 180-182 of SEQ ID NO:1). The resulting secreted factor Xzymogen consists of an N-terminal light chain of 139 amino acids and aC-terminal heavy chain of 306 amino acids which are covalently linkedvia a disulfide bridge between Cys132 and Cys302. As used herein, theterm “activated Factor X” or “FXa” refers to the enzymatically activeform of circulating factor X generated in case of coagulation activity(e.g. thrombin generation) is needed. Under physiological conditionsable to activate factor X, the so called activation peptide of 52 aminoacids from Ser143 to Arg194 is cleaved off the rest of the molecule bycleaving carboxy-terminal end of the heavy chain at Arg194 (FIG. 1).According to the numbering system of the present invention, thepositions 215, 216 and 218 in Factor X corresponds respectively topositions 255, 256 and 258 in SEQ ID NO: 1.

As used herein, the term “substitution” means that a specific amino acidresidue at a specific position is removed and another amino acid residueis inserted into the same position.

In some embodiments, the present invention relates to a mutated factor X(FX) polypeptide wherein the heavy chain comprises at least one mutationwherein the glutamic acid residue (E) at position 215, 216 or 218 issubstituted by a glutamine residue (Q).

In some embodiments, the present invention relates to a mutated factor X(FX) polypeptide wherein the heavy chain comprises one mutation whereinthe glutamic acid residue (E) at position 215 is substituted by aglutamine residue (Q).

In some embodiments, the present invention relates to a mutated factor X(FX) polypeptide wherein the heavy chain comprises one mutation whereinthe glutamic acid residue (E) at position 216 is substituted by aglutamine residue (Q).

In some embodiments, the present invention relates to a mutated factor X(FX) polypeptide wherein the heavy chain comprises one mutation whereinthe glutamic acid residue (E) at position 218 is substituted by aglutamine residue (Q).

In some embodiments, the mutated FX polypeptide of the present inventioncomprises a heavy chain which consists of the amino acid sequence havingat least 90% of identity with the sequence ranging from the amino acidresidue at position 195 to the amino acid residue at position 448wherein at least one glutamic acid residue at position 215, 216 or 218is mutated according to the invention. According to the invention afirst amino acid sequence having at least 90% of identity with a secondamino acid sequence means that the first sequence has 90; 91; 92; 93;94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acidsequence. In some embodiments, the mutated FX polypeptide of theinvention comprises a heavy chain which consists of the amino acidsequence ranging from the amino acid residue at position 195 to theamino acid residue at position 448 wherein at least one glutamic acidresidue at position 215, 216 or 218 is mutated according to theinvention.

In some embodiments, the mutated FX polypeptide of the present inventioncomprises a heavy chain wherein the amino acid at position 196 is notsubstituted.

In some embodiments, the mutated FX polypeptide of the present inventionfurther comprises a sequence cleavable by thrombin. In some embodiments,three amino acid of the activation peptide of FX (Thr-Arg-Ile) aresubstituted by Pro-Arg-Ala, thus, the mutated FX polypeptide constitutesa chimeric thrombin-cleavable derivative of factor X (as described inWO04005347). In some embodiments, the activation peptide of FX issubstituted by fibrinopeptide A sequence, thus the mutated FXpolypeptide constitutes a chimeric thrombin-cleavable derivative offactor X (as described in WO03035861). In some embodiments, thefibrinopeptide A sequence is inserted between the activation peptide andthe heavy chain so that the mutated FX polypeptide constitutes achimeric thrombin-cleavable derivative of factor X (as described inWO2010070137). As used herein, the term “fibrinopeptide A” has itsgeneral meaning in the art and refers to a small peptide of 16 aminoacid residues removed from the N-terminal segment of the α-chain offibrinogen by the action of thrombin. The amino acid sequence of thehuman fibrinopeptide A is provided by SEQ ID NO:2.

According to the invention, the mutated FX polypeptide of the inventionis produced by conventional automated peptide synthesis methods or byrecombinant expression. General principles for designing and makingproteins are well known to those of skill in the art. The mutated FXpolypeptides of the invention may be synthesized in solution or on asolid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols as described in Stewart and Young; Tamet al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross andMeienhofer, 1979. The mutated FX polypeptides of the invention may alsobe synthesized by solid-phase technology employing an exemplary peptidesynthesizer such as a Model 433A from Applied Biosystems Inc. The purityof any given protein; generated through automated peptide synthesis orthrough recombinant methods may be determined using reverse phase HPLCanalysis. Chemical authenticity of each peptide may be established byany method well known to those of skill in the art. As an alternative toautomated peptide synthesis, recombinant DNA technology may be employedwherein a nucleotide sequence which encodes a protein of choice isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression as described herein below. Recombinant methods are especiallypreferred for producing longer polypeptides. A variety of expressionvector/host systems may be utilized to contain and express the peptideor protein coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors (Giga-Hama et al., 1999);insect cell systems infected with virus expression vectors (e.g.,baculovirus, see Ghosh et al., 2002); plant cell systems transfectedwith virus expression vectors (e.g., cauliflower mosaic virus, CaMV;tobacco mosaic virus, TMV) or transformed with bacterial expressionvectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); oranimal cell systems. Those of skill in the art are aware of varioustechniques for optimizing mammalian expression of proteins, see e.g.,Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful inrecombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of thepeptide substrates or fusion polypeptides in bacteria, yeast and otherinvertebrates are known to those of skill in the art and a brieflydescribed herein below. Mammalian host systems for the expression ofrecombinant proteins also are well known to those of skill in the art.Host cell strains may be chosen for a particular ability to process theexpressed protein or produce certain post-translation modifications thatwill be useful in providing protein activity. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, andthe like have specific cellular machinery and characteristic mechanismsfor such post-translational activities and may be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

In specific embodiments, it is contemplated that the mutated FXpolypeptides of the invention used in the therapeutic methods of thepresent invention may be modified in order to improve their therapeuticefficacy. Such modification of therapeutic compounds may be used todecrease toxicity, increase circulatory time, or modify biodistribution.For example, the toxicity of potentially important therapeutic compoundscan be decreased significantly by combination with a variety of drugcarrier vehicles that modify biodistribution. A strategy for improvingdrug viability is the utilization of water-soluble polymers. Variouswater-soluble polymers have been shown to modify biodistribution,improve the mode of cellular uptake, change the permeability throughphysiological barriers; and modify the rate of clearance from the body.To achieve either a targeting or sustained-release effect, water-solublepolymers have been synthesized that contain drug moieties as terminalgroups, as part of the backbone, or as pendent groups on the polymerchain. Polyethylene glycol (PEG) has been widely used as a drug carrier,given its high degree of biocompatibility and ease of modification.Attachment to various drugs, proteins, and liposomes has been shown toimprove residence time and decrease toxicity. PEG can be coupled toactive agents through the hydroxyl groups at the ends of the chain andvia other chemical methods; however, PEG itself is limited to at mosttwo active agents per molecule. In a different approach, copolymers ofPEG and amino acids were explored as novel biomaterials which wouldretain the biocompatibility properties of PEG, but which would have theadded advantage of numerous attachment points per molecule (providinggreater drug loading), and which could be synthetically designed to suita variety of applications.

A further object of the present invention relates to a nucleic acidmolecule which encodes for a mutated FX polypeptide of the presentinvention.

As used herein, the term “nucleic acid molecule” has its general meaningin the art and refers to a DNA or RNA molecule. However, the termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiments, the nucleic acid molecule of the present inventionis included in a suitable vector, such as a plasmid, cosmid, episome,artificial chromosome, phage or a viral vector. So, a further object ofthe invention relates to a vector comprising a nucleic acid encoding fora mutated FX polypeptide of the invention. Typically, the vector is aviral vector which is an adeno-associated virus (AAV), a retrovirus,bovine papilloma virus, an adenovirus vector, a lentiviral vector, avaccinia virus, a polyoma virus, or an infective virus. In someembodiments, the vector is an AAV vector. As used herein, the term “AAVvector” means a vector derived from an adeno-associated virus serotype,including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or moreof the AAV wild-type genes deleted in whole or part, preferably the repand/or cap genes, but retain functional flanking ITR sequences.Retroviruses may be chosen as gene delivery vectors due to their abilityto integrate their genes into the host genome, transferring a largeamount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and for being packaged in special cell-lines. Inorder to construct a retroviral vector, a nucleic acid encoding a geneof interest is inserted into the viral genome in the place of certainviral sequences to produce a virus that is replication-defective. Inorder to produce virions, a packaging cell line is constructedcontaining the gag, pol, and/or env genes but without the LTR and/orpackaging components. When a recombinant plasmid containing a cDNA,together with the retroviral LTR and packaging sequences is introducedinto this cell line (by calcium phosphate precipitation for example),the packaging sequence allows the RNA transcript of the recombinantplasmid to be packaged into viral particles, which are then secretedinto the culture media. The media containing the recombinantretroviruses is then collected, optionally concentrated, and used forgene transfer. Retroviral vectors are able to infect a broad variety ofcell types. Lentiviruses are complex retroviruses, which, in addition tothe common retroviral genes gag, pol, and env, contain other genes withregulatory or structural function. The higher complexity enables thevirus to modulate its life cycle, as in the course of latent infection.Some examples of lentivirus include the Human Immunodeficiency Viruses(HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviralvectors have been generated by multiply attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu and nef are deletedmaking the vector biologically safe. Lentiviral vectors are known in theart, see, e.g. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which areincorporated herein by reference. In general, the vectors areplasmid-based or virus-based, and are configured to carry the essentialsequences for incorporating foreign nucleic acid, for selection and fortransfer of the nucleic acid into a host cell. The gag, pol and envgenes of the vectors of interest also are known in the art. Thus, therelevant genes are cloned into the selected vector and then used totransform the target cell of interest. Recombinant lentivirus capable ofinfecting a non-dividing cell wherein a suitable host cell istransfected with two or more vectors carrying the packaging functions,namely gag, pol and env, as well as rev and tat is described in U.S.Pat. No. 5,994,136, incorporated herein by reference. This describes afirst vector that can provide a nucleic acid encoding a viral gag and apol gene and another vector that can provide a nucleic acid encoding aviral env to produce a packaging cell. Introducing a vector providing aheterologous gene into that packaging cell yields a producer cell whichreleases infectious viral particles carrying the foreign gene ofinterest. The env preferably is an amphotropic envelope protein whichallows transduction of cells of human and other species. Typically, thenucleic acid molecule or the vector of the present invention include“control sequences”, which refers collectively to promoter sequences,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control sequences need always bepresent so long as the selected coding sequence is capable of beingreplicated, transcribed and translated in an appropriate host cell.Another nucleic acid sequence, is a “promoter” sequence, which is usedherein in its ordinary sense to refer to a nucleotide region comprisinga DNA regulatory sequence, wherein the regulatory sequence is derivedfrom a gene which is capable of binding RNA polymerase and initiatingtranscription of a downstream (3′-direction) coding sequence.Transcription promoters can include “inducible promoters” (whereexpression of a polynucleotide sequence operably linked to the promoteris induced by an analyte, cofactor, regulatory protein, etc.),“repressible promoters” (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), and “constitutive promoters”.

A further object of the present invention relates to a host celltransformed with the nucleic acid molecule of the present invention. Theterm “transformation” means the introduction of a “foreign” (i.e.extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, sothat the host cell will express the introduced gene or sequence toproduce a desired substance, typically a protein or enzyme coded by theintroduced gene or sequence. A host cell that receives and expressesintroduced DNA or RNA has been “transformed”. In a particularembodiment, for expressing and producing the mutated FX polypeptides ofthe present invention, prokaryotic cells, in particular E. Coli cells,will be chosen. Actually, according to the invention, it is notmandatory to produce the mutated FX polypeptides of the presentinvention in a eukaryotic context that will favour post-translationalmodifications (e.g. glycosylation). Furthermore, prokaryotic cells havethe advantages to produce protein in large amounts. If a eukaryoticcontext is needed, yeasts (e.g. saccharomyces strains) may beparticularly suitable since they allow production of large amounts ofproteins. Otherwise, typical eukaryotic cell lines such as CHO, BHK-21,COS-7, C127, PER.C6, YB2/0 or HEK293 could be used, for their ability toprocess to the right post-translational modifications of the mutated FXpolypeptides of the present invention. The construction of expressionvectors in accordance with the invention, and the transformation of thehost cells can be carried out using conventional molecular biologytechniques. The mutated FX polypeptide of the invention, can, forexample, be obtained by culturing genetically transformed cells inaccordance with the invention and recovering the mutated FX polypeptideexpressed by said cell, from the culture. They may then, if necessary,be purified by conventional procedures, known in themselves to thoseskilled in the art, for example by fractional precipitation, inparticular ammonium sulfate precipitation, electrophoresis, gelfiltration, affinity chromatography, etc. In particular, conventionalmethods for preparing and purifying recombinant proteins may be used forproducing the proteins in accordance with the invention.

The mutated FX polypeptides of the present invention and nucleic acidmolecules encoding thereof are typically used as medicament. Inparticular, the nucleic acid molecules of the present invention(inserted or not into a vector) are particularly suitable for genetherapy. In some embodiments, the mutated FX polypeptides of the presentinvention and nucleic acid molecules (inserted or not into a vector) areparticularly suitable for the treatment of haemophilia. Haemophiliaincludes haemophilias A or B, which may or may not be complicated by thepresence of inhibitors (neutralizing allo-antibodies directed againstthe factor VIII or IX conventionally used for treatment); they may alsobe acquired haemophilias resulting from the appearance of autoantibodies associated with another pathology (autoimmune disease,cancer, lymphoproliferative syndrome, idiopathic disorder, etc.).

Accordingly a further object of the present invention relates to amethod for treating haemophilia in a subject in need thereof comprisingadministering the subject with a therapeutically effective amount of amutated FX polypeptide of the present invention or a nucleic acidmolecule of the present invention which is inserted or not in to avector as above described.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By “therapeutic regimen” is meant the pattern oftreatment of an illness, e.g., the pattern of dosing used duringtherapy. A therapeutic regimen may include an induction regimen and amaintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a patient during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer the drug during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a patientduring treatment of an illness, e.g., to keep the patient in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at a regularintervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy(e.g., interrupted treatment, intermittent treatment, treatment atrelapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., disease manifestation, etc.]).

By a “therapeutically effective amount” is meant a sufficient amount ofthe mutated FX polypeptide or the nucleic acid molecule encoding thereofto prevent for use in a method for the treatment of the disease (e.g.haemophilia) at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood that the total daily usage ofthe compounds and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the age, bodyweight, general health, sex and diet of the subject; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific polypeptide employed; andlike factors well known in the medical arts. For example, it is wellknown within the skill of the art to start doses of the compound atlevels lower than those required to achieve the desired therapeuticeffect and to gradually increase the dosage until the desired effect isachieved. However, the daily dosage of the products may be varied over awide range from 0.01 to 1,000 mg per adult per day. Preferably, thecompositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, 50.0, 100, 250 and 500 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject to be treated. Amedicament typically contains from about 0.01 mg to about 500 mg of theactive ingredient, preferably from 1 mg to about 100 mg of the activeingredient. An effective amount of the drug is ordinarily supplied at adosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day,especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

According to the invention, the mutated FX polypeptide or the nucleicacid molecule (inserted or not into a vector) of the present inventionis administered to the subject in the form of a pharmaceuticalcomposition. Typically, the mutated FX polypeptide or the nucleic acidmolecule (inserted or not into a vector) of the present invention may becombined with pharmaceutically acceptable excipients, and optionallysustained-release matrices, such as biodegradable polymers, to formpharmaceutical compositions. “Pharmaceutically” or “pharmaceuticallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to a mammal, especially a human, as appropriate. Apharmaceutically acceptable carrier or excipient refers to a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. In the pharmaceutical compositions ofthe present invention for oral, sublingual, subcutaneous, intramuscular,intravenous, transdermal, local or rectal administration, the activeprinciple, alone or in combination with another active principle, can beadministered in a unit administration form, as a mixture withconventional pharmaceutical supports, to animals and human beings.Suitable unit administration forms comprise oral-route forms such astablets, gel capsules, powders, granules and oral suspensions orsolutions, sublingual and buccal administration forms, aerosols,implants, subcutaneous, transdermal, topical, intraperitoneal,intramuscular, intravenous, subdermal, transdermal, intrathecal andintranasal administration forms and rectal administration forms.Typically, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions. The pharmaceutical forms suitablefor injectable use include sterile aqueous solutions or dispersions;formulations including sesame oil, peanut oil or aqueous propyleneglycol; and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Solutions comprisingcompounds of the invention as free base or pharmacologically acceptablesalts can be prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. The mutated FXpolypeptide or the nucleic acid molecule (inserted or not into a vector)of the present invention can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. The carrier can also be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin. Sterile injectable solutions are prepared byincorporating the active compounds in the required amount in theappropriate solvent with several of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the typical methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparation of more, or highlyconcentrated solutions for direct injection is also contemplated, wherethe use of DMSO as solvent is envisioned to result in extremely rapidpenetration, delivering high concentrations of the active agents to asmall tumor area. Upon formulation, solutions will be administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed. For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: Schematic representation of different parts of factor X zymogenamino acid sequence. The pre-peptide (or signal peptide) is defined bythe amino acid sequence between the positions −40 to −18 and thepro-peptide by the amino acid sequence between the positions −17 to −1.The light chain corresponds to the sequence between the amino acidpositions 1 to 142 and the heavy chain between amino acid positions 195to 448. The activation peptide (positions 143 to 194) is boxed andN-glycosylation sites of interest are tagged by an *. The numberingsystem used appears on the same line as the sequence and the otherreference system appears in grey on the line under the sequence.

EXAMPLE

Material & Methods

Engineering and Production of Recombinant FX and FX Derivatives

cDNAs encoding wild-type (wt)-human FX (wt-hFX), and its variantsFXE215Q, FXE216Q and FXE218Q (FIG. 1) were synthetically synthesized(Eurofins, Ebersberg Germany) and cloned in expression vectors usingstandard molecular biological protocols. Constructs were cloned into thepLIVE- and pNUT-plasmids for in vivo expression and in vitro expressionin stably transfected in BHK-21 cells, respectively (23, 24, 25). Allconstructs contained a DNA sequence encoding the epitope recognized byantibody HPC4 (Roche, Meylan, France) that was added at the 3′ end ofthe FX sequence. Stable BHK-21 cell lines producing FX or variantsthereof were established as described (25) and detailed in the followingsection.

Obtention of Cell Lines Expressing the Recombinant Derivatives

The pNUT-constructs were transfected into Baby hamster kidney cells(BHK) using the jetPEI reactant (Qbiogen, Ozyme, France) as specified bythe provider. After selection of transfected cells with mediumcontaining methotrexate (Sigma) at a concentration of 100 μM, singleclones were picked and propagated in selective medium to obtain stablecell lines. Production of factor X antigen was assayed by enzyme-linkedimmunosorbent assay (ELISA) using polyclonal antibodies against factor Xconjugated and not with horseradish peroxidase obtained from Cederlane(Cederlane Laboratories, Burlington, Canada). Purified human plasmaderived factor X (pd-FX) from Cryopep (Montepellier, France) was used asreference.

Production and Purification of Recombinant Factor X and Derivatives

Stable cell lines producing recombinant factor X, and were maintained in300 cm2 flasks for protein production in DMEM/F-12 supplemented with 10%FCS, 50 μM methotrexate, 100 U/ml penicillin, 100 μg/ml streptomycin,and 5 μg/ml vitamin K1. Protein of interest containing medium washarvested every 48 hours. Benzamidine and PMSF were added to a finalconcentration of 10 and 2 mM, respectively, and the medium centrifuged(6 000 g), passed over cellulose acetate membranes (0.45 μm) toeliminate cell debris, and stored at −20° C. until use. Conditionedmedium was thawed at 37° C. EDTA was added to a final concentration of 5mM. The medium was diluted in distilled water and in Tris (pH 7.4), tobring the final Tris and NaCl concentration to 25 and 60 mM,respectively. The mixture was then stirred at room temperature for 30min with QAE Sephadex A-50 beads to achieve a final concentration of0.25% (wt/v). Beads were washed before elution with 50 mM Tris (pH 7.4),500 mM NaCl, and 10 mM benzamidine. Recombinant proteins contained inthe eluted fractions (ELISA) were immediately dialyzed against 25 mMTris (pH 7.4), and 100 mM NaCl, containing 10 mM benzamidine, and storedat −20° C. before use. Concentrated proteins were thawed at 37° C.Calcium was added to a final concentration of 5 mM. Purification ofrecombinant proteins was performed by affinity-chromatography usingHPC-4-agarose (Roche, Meylan, France) as instructed by the provider. 1 hprior to use as a zymogen, factor X derivatives were incubated with 1 mMPMSF to neutralize any trace of activated factor X that may have beengenerated during production or purification of the recombinant protein.Control experiments indicated that after 30 min in Tris-HCl buffer, PMSFwas fully hydrolyzed and would not interfere with other reactions.Protein purity was assessed using 10% SDS-polyacrylamide gelelectrophoresis analysis of the recombinant proteins under reducing (100mM dithiothreitol, final concentration) and non-reducing conditionsfollowed by staining with Coomassie Brilliant Blue R-250. Factor Xidentification was carried out after the purified recombinant proteinswere reduced and loaded onto a 10% SDS-polyacrylamide gel. The resolvedproteins were transferred to an Immobilon membrane and blotted usingpolyclonal antibodies against factor X conjugated with horseradishperoxydase (Cederlane). The purified derivatives were aliquoted andstored at −80° C. until use. The concentration of the aliquot isestimated by its absorbance at 280 nm, taking 1.16 to be the extinctioncoefficient (E280 nm 0.1%) of factor X.

Thrombin Generation Assay

Thrombin generation was measured according to the method described byHemker et al (26), in a Fluoroscan Ascent fluorometer (ThermolabsystemsOY, Helsink, Finland) equipped with a dispenser. Briefly, 80 μl ofplasma supplemented with either saline (control) or with indicatedconcentration of recombinant factor X derivatives were dispensed intoround-bottom 96-well microtiter plates. Twenty μ1 of a mixturecontaining TF (recombinant lipidated human tissue factor, Innovin®,obtained from Dade Behring) and phospholipids (PL) vesicles was added tothe plasma sample to obtain a final concentration of 1 pM TF and 4 μM PLvesicles. PL vesicles prepared from L-α-Phosphatidyl-L-serine (PS)L-α-phosphatidylethanolamine (PE) and L-α-phosphatidylcholine (PC)(Avanti Polarlipids, Alabaster, Ala., USA) and of nominal 100nm-diameter (PC:PE:PS, 3:1:1) were synthesized by the method of membraneextrusion (27). Phospholipid concentration was determined by phosphateanalysis. Finally, thrombin generation was triggered by adding 20 μl ofstarting reagent containing fluorogenic substrate and CaCl₂. Fluorogenicsubstrate 1-1140 (Z-Gly-Gly-Arg-AMC) was from Bachem AG (Bubendorf,Switzerland). Kinetics of thrombin generation in clotting plasma wasmonitored for 60 min at 37° C. using a calibrated automated thrombogramand analyzed using the Thrombinoscope™ software (Thrombinoscope B.V.,Maastricht, the Netherlands). Four wells were needed for eachexperiment, two wells to measure thrombin generation of a plasma sampleand two wells for calibration. All experiments were carried out intriplicate and the mean value was reported. Endogenous thrombinpotential (ETP), i.e. area under the curve, peak thrombin, and lag timefor thrombin detection determined. In some experiments, immunodepletedFVIII-plasma (Diagnostica Stago, Asnieres, France) was supplemented withFX variants (60, 150 and 300 nM final concentrations) or recombinantpurified FVIII (0.025, 0.1, and 1 U/ml Kogenate® FS, Bayer HealthCare,Puteaux, France). In other experiments, immunodepleted FVIII-deficienthuman plasma was supplemented with mouse monoclonal anti-FVIII antibodyD4H1 to create FVIII-inhibitor plasma (28). D4H1 at a finalconcentration of 10 μg/ml or 50 μg/ml corresponds to 30-40 BethesdaUnits (BU)/ml and 150-200 BU, respectively. Subsequently, FVIIIinhibitor plasma was supplemented in vitro with various concentrationsFX derivatives. Finally, experiments were performed using immunodepletedFIX-plasma (Diagnostica Stago, Asnieres, France).

Results:

Thrombin Generation in FVIII and FIX-Deficient Plasmas

In a first series of experiments, we assessed the potential of differentconcentrations of FVIII, 1, 0.1, and 0.025 U/ml corresponding to anormal individual (control), a mild and a moderate hemophilia,respectively, to compensate for the absence of FVIII in the generationof thrombin. To this end, coagulation in immunodepleted FVIII-deficienthuman plasma was initiated by the addition of TF (1 pM) andphospholipids (4 μM), and relevant thrombin generation parameters suchas ETP and peak thrombin generation were determined. In the absence ofany added coagulation factor, this resulted in an ETP and a peakthrombin generation (for summary see Table 1). Both values aresignificantly reduced compared to normal plasma (ETP: ˜1200 nM·min; peakthrombin generation: 150-174 nM). As expected, the addition of FXderivatives (60, 150, 200 and 300 nM final concentrations) resulted inrestoration of thrombin generation in FVIII-deficient plasma (Table 1).Also the addition of FXE215Q, FXE216Q or FXE218Q to a concentration of300 nM resulted in normalization of thrombin generation, with both ETPand peak thrombin generation being within the same range as found fornormal plasma (Table 1). A similar correction of the coagulation defectwas observed when tested in immunodepleted FIX-deficient plasma (Table2). Furthermore, no correction of thrombin generation was observed bythe addition of wt-FX up to the highest concentration tested (0.5 μM).These data indicate that under the conditions employed, the presence ofthe mutation gives the capacity to FX to overcome the absence of FVIIIor FIX for efficient thrombin generation.

Thrombin Generation in FVIII-Deficient Inhibitor Plasma

We next evaluated FX derivatives for their ability to correct thecoagulation deficiency in FVIII-deficient plasma in the presence ofanti-FVIII antibody 4D1, dosed at a concentration of 150 BU/ml. Theaddition of increasing concentration FX derivatives resulted innormalization of the total thrombin generation (Table 3). Thus, theFXE215Q, FXE216Q or FXE218Q appears to be an efficient pro-coagulantagent to correct thrombin generation in FVIII-inhibitor plasma.

Tables:

TABLE 1 Thrombin generation test in FVIII-deficient plasma (Cryopep,Montpellier). Parameters for measuring thrombin generation (ETP,thrombin peak) were measured in immunodepleted FVIII-deficient plasma inthe presence of tissue factor (1 pM) and phospholipids (4 μM) with orwithout FVIII, FXE215Q, FXE216Q or FXE218Q. Data are presented as mean ±SD. Added coagulation factor in FVIII-deficient ETP Thromin Peak plasman (nM · min) (nM) FVIII (1 U/ml) 7 815 ± 52 179 ± 8  FVIII (0.1 U/ml) 6634 ± 31 56 ± 5 FVIII (0.025 U/ml) 6 441 ± 38 28 ± 3 None 7 213 ± 27 11± 1 FXE215Q (300 nM) 3 835 ± 35 156 ± 7  FXE215Q (150 nM) 3 762 ± 96 66± 5 FXE215Q (60 nM) 3 558 ± 43 39 ± 2 FXE216Q (200 nM) 3 795 ± 25 119 ±5  FXE218Q (300 nM) 3 856 ± 40 139 ± 4  FXE218Q (150 nM) 3 771 ± 45 58 ±4 FXE218Q (60 nM) 3 297 ± 28 18 ± 1

TABLE 2 Thrombin generation test in FIX-immunodepleted plasma (Stago,France). Parameters for measuring thrombin generation (ETP, thrombinpeak) were measured in immunodepleted FIX-deficient plasma in thepresence of tissue factor (1 pM) and phospholipids (4 μM) with orwithout FIX, FXE215Q, FXE216Q or FXE218Q. Data are presented as mean ±SD. Added coagulation factor in FIX-deficient ETP Thromin Peak plasma n(nM · min) (nM) FIX (1 U/ml) 3 1356 ± 30  295 ± 8  FIX (0.1 U/ml) 3 802± 19 81 ± 5 FIX (0.025 U/ml) 3 424 ± 18 29 ± 1 None 3 239 ± 68 10 ± 3FXE215Q 3 1019 ± 17  223 ± 3  (300 nM) FXE215Q (150 nM) 3 892 ± 30 49 ±4 FXE215Q (60 nM) 3 467 ± 12 21 ± 4 FXE216Q (200 nM) 3 787 ± 22 121 ± 5 FXE218Q (300 nM) 3 1188 ± 17  294 ± 8  FXE218Q (150 nM) 3 993 ± 23 81 ±4 FXE218Q (60 nM) 3 491 ± 20 29 ± 4

TABLE 3 Thrombin generation test in FVIII-deficient plasma (Cryopep,Montpellier) in the presence of inhibitor. Parameters for measuringthrombin generation (ETP, thrombin peak) were measured inFVIII-deficient plasma supplemented with mouse monoclonal anti-FVIIIantibody D4H1 to create FVIII-inhibitor plasma in the presence of tissuefactor (1 pM) and phospholipids (4 μM) with or without FVIII, FXE215Q,FXE216Q or FXE218Q. Data are presented as mean ± SD. Added coagulationfactor in FVIII-deficient plasma supplemented with ETP Thromin Peakinhibitor n (nM · min) (nM) FXE215Q (300 nM) 3 978 ± 41 101 ± 6  FXE215Q(150 nM) 3 536 ± 20 48 ± 4 FXE215Q (60 nM) 3 306 ± 15 17 ± 4 FXE215Q (30nM) 3 208 ± 11  8 ± 4 FXE216Q (200 nM) 3 685 ± 20 54 ± 4 FXE218Q (300nM) 3 949 ± 41 113 ± 4  FXE218Q (150 nM) 3 643 ± 11 64 ± 4 FXE218Q (60nM) 3 418 ± 9  24 ± 2 FXE218Q (30 nM) 3 276 ± 13 14 ± 1

SEQUENCES

SEQ ID NO: 1: Factor X (homo sapiens)MGRPLHLVLL SASLAGLLLL GESLFIRREQ ANNILARVTRANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEFWNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKNCELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADNGKACIPTGPY PCGKQTLERR KRSVAQATSS SGEAPDSITWKPYDAADLDP TENPFDLLDF NQTQPERGDN NLTRIVGGQECKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQAKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYDFDIAVLRLKT PITFRMNVAP ACLPERDWAE STLMTQKTGIVSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQNMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE VITSSPLKSEQ ID NO: 2: fibrinopeptide A (homo sapiens) ADSGEGDFLA EGGGVR

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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1. A mutated factor X (FX) polypeptide comprising a heavy chaincomprising at least one mutation selected from the group consisting of:a first mutation comprising substitution of a glutamic acid residue (E)at position 215 by a glutamine residue (Q), an asparagine residue (N), aserine residue (S), an alanine residue (A), or a tyrosine residue (Y); asecond mutation comprising substitution of a glutamic acid residue (E)at position 216 by a glutamine residue (Q); and/or a third mutationcomprising substitution of a glutamic acid residue (E) at position 218by a glutamine residue (Q).
 2. The mutated factor X (FX) polypeptide ofclaim 1 wherein the heavy chain comprises at least one mutation whereinthe glutamic acid residue (E) at position 215, 216 or 218 is substitutedby a glutamine residue (Q).
 3. The mutated factor X (FX) polypeptide ofclaim 1 wherein the heavy chain comprises at least one mutation whereinthe glutamic acid residue (E) at position 215 is substituted by aglutamine residue (Q).
 4. The mutated factor X (FX) polypeptide of claim1 wherein the heavy chain comprises at least one mutation wherein theglutamic acid residue (E) at position 216 is substituted by a glutamineresidue (Q).
 5. The mutated factor X (FX) polypeptide of claim 1 whereinthe heavy chain comprises at least one mutation wherein the glutamicacid residue (E) at position 218 is substituted by a glutamine residue(Q).
 6. The mutated factor X (FX) polypeptide of claim 1 which comprisesa heavy chain comprising an amino acid sequence having at least 90%identity with a sequence ranging from an amino acid residue at position195 to an amino acid residue at position
 448. 7. The mutated factor X(FX) polypeptide of claim 1 which comprises a heavy chain wherein anamino acid at position 196 is not substituted.
 8. The mutated factor X(FX) polypeptide of claim 1 which further comprises a fibrinopeptide Awhich is inserted between an activation peptide and the heavy chain. 9.The mutated factor X (FX) polypeptide of claim 8 wherein thefibrinopeptide A comprises the amino acid sequence of SEQ ID NO:2.
 10. Anucleic acid molecule which encodes for the mutated factor X (FX)polypeptide of claim
 1. 11. A vector which comprises the nucleic acidmolecule of claim
 10. 12. A host cell which is transformed with thenucleic acid molecule of claim 10 or a vector comprising the nucleicacid.
 13. (canceled)
 14. A method of treating haemophilia in a subjectin need thereof comprising administering to the subject atherapeutically effective amount of the mutated factor X (FX)polypeptide of claim 1 or a nucleic acid molecule encoding the mutatedfactor X (FX) polypeptide.
 15. A pharmaceutical composition whichcomprises the mutated factor X (FX) polypeptide of claim 1 or a nucleicacid molecule encoding the mutated factor X (FX) polypeptide.
 16. Themethod of claim 14, wherein the nucleic acid molecule is present in avector.
 17. The method of claim 14, wherein the nucleic acid molecule isnot present in a vector.